Liposomal Formulations of a Polyleucine–Antigen Conjugate as Therapeutic Vaccines against Cervical Cancer

Human papilloma virus (HPV) is responsible for all cases of cervical cancer. While prophylactic vaccines are available, the development of peptide-based vaccines as a therapeutic strategy is still under investigation. In comparison with the traditional and currently used treatment strategies of chemotherapy and surgery, vaccination against HPV is a promising therapeutic option with fewer side effects. A peptide derived from the HPV-16 E7 protein, called 8Qm, in combination with adjuvants showed promise as a therapeutic vaccine. Here, the ability of polymerized natural amino acids to act as a self-adjuvating delivery system as a therapeutic vaccine was investigated for the first time. Thus, 8Qm was conjugated to polyleucine by standard solid-phase peptide synthesis and self-assembled into nanoparticles or incorporated in liposomes. The liposome bearing the 8Qm conjugate significantly increased mice survival and decreased tumor growth after a single immunization. Further, these liposomes eradicated seven-day-old well-established tumors in mice. Dendritic cell (DC)-targeting moieties were introduced to further enhance vaccine efficacy, and the newly designed liposomal vaccine was tested in mice bearing 11-day-old tumors. Interestingly, these DCs-targeting moieties did not significantly improve vaccine efficacy, whereas the simple liposomal formulation of 8Qm-polyleucine conjugate was still effective in tumor eradication. In summary, a peptide-based anticancer vaccine was developed that stimulated strong cellular immune responses without the help of a classical adjuvant.


Introduction
Over 600 thousand people are diagnosed with cervical cancer each year triggered by the human papilloma virus (HPV), and 342 thousand people died worldwide as a result of the disease in 2020 alone [1]. Over the past few decades, the number of HPVrelated cancer cases has risen, especially in developing countries. While early identification through comprehensive cervical screening programs has decreased the prevalence of cervical cancer, these resources are less accessible in under-developed countries. Treatment options currently available for cervical cancer include chemotherapy, radiotherapy and surgery. However, these treatments have a high failure rate due to significant relapse rates and the development of drug resistance. A prophylactic vaccine has been developed against HPV infection, which will likely decrease the overall rate of cervical cancer; however, conjugates anchored to DC-targeting liposomes were expected to demonstrate improved anticancer efficacy.
The aim of this study was to develop a new antitumor vaccine based on a novel polymeric/liposomal delivery system and demonstrate its efficacy against cervical cancer. pLeu was attached to the 8Qm peptide to form pLeu-8Qm, and it was then anchored into a secondary delivery system involving liposomes. A variety of dendritic cell (DC)-targeting moieties were further incorporated into these liposomes to potentially enhance the vaccine's efficacy ( Figure 1). as monomers may overcome all disadvantages of classic polymer-based delivery systems. We have demonstrated that a pHAA sequence consisting of pLeu conjugated to the J8 Bcell epitope from group A Streptococcus self-assembled into nanoparticles stimulated the production of high titers of opsonic antibodies in mice [23,24]. Moreover, pLeu-based peptide vaccines were very effective in triggering humoral immunity against hookworm and in parasite clearance from infected animals [25][26][27]. Here, we hypothesize that pLeu can be also employed in a conjugation-based delivery system to induce cytotoxic T-lymphocyte (CTL) responses. Moreover, pLeu conjugates anchored to DC-targeting liposomes were expected to demonstrate improved anticancer efficacy.
The aim of this study was to develop a new antitumor vaccine based on a novel polymeric/liposomal delivery system and demonstrate its efficacy against cervical cancer. pLeu was attached to the 8Qm peptide to form pLeu-8Qm, and it was then anchored into a secondary delivery system involving liposomes. A variety of dendritic cell (DC)-targeting moieties were further incorporated into these liposomes to potentially enhance the vaccine's efficacy (Figure 1).

Formulation of Vaccine Candidates
Formulation of L1 D-8Qm and liposomes L1 were formulated as previously reported [19]. Formulation of pLeu-8Qm pLeu-8Qm was dissolved in Millipore endotoxin-free water (900 µL) and self-assembled into nanoparticles. Prior to injection, PBS (100 µL, 10×) was added to the formulation, producing the final concentration of 1 mg/mL.

Formulation of L2
Dipalmitoylphosphatidylcholine (DPPC), didodecyldimethylammonium bromide (DDAB) and cholesterol were each dissolved in chloroform (1 mL) to achieve final concentrations of 10 mg/mL, 10 mg/mL and 5 mg/mL, respectively. Solutions of DPPC (0.5 mL), DDAB (0.2 mL) and cholesterol (0.2 mL) were added to a round-bottom flask containing chloroform (2 mL). pLeu-8Q (1 mg) was dissolved in methanol (1 mL) and added to the flask. The solvents were then slowly evaporated under reduced pressure using a rotatory evaporator before the residual solvent was removed under vacuum overnight generating a lipid film. The lipid film was rehydrated with Millipore endotoxin-free water (900 µL) at 56 • C following extrusion with 200 nm pore-size polycarbonate membrane. Prior to injection, PBS (100 µL, 10×) was added to the formulation, producing the final concentration of pLeu-8Qm (1 mg/mL) in L2. The encapsulation efficiency of pLeu-8Qm in liposomes was 99%. Encapsulation was determined as previously reported [19].

Formulation of L2CPP
The liposomes bearing targeting moieties were formulated in a similar manner to L2, except DC-targeting moieties CPP (0.25 mg)-added along with pLeu-8Q to the lipid solution. In addition, DDAB was excluded from lipid solution. Lipid film was rehydrated with 900 µL of Millipore endotoxin-free water at 56 • C and not extruded. Finally, 100 µL of 10× PBS was added to the formulation, producing the final concentration of pLeu-8Qm (1 mg/mL) as multilamellar liposomes.

Characterization of Nanoliposomes
Particle size, zeta potential and polydispersity of the vaccine candidates were measured by dynamic light scattering (DLS) using a Zetasizer (Nano ZX, Marvern, UK). Measurements were taken at 25 • C and 173 • light scattering. DLS measurements were taken using a Zetasizer Nano ZP instrument (Malvern Instrument, UK) with Malvern Zetasizer Analyzer 6.2 software. Particle morphology was examined using transmission electron microscopy (TEM) (HT7700 Exalens, HITACHI Ltd., Tokyo, Japan) after vacuum drying. Briefly, samples were diluted in pure distilled water (1:100), dropped directly onto glowdischarged carbon-coated copper grids, then stained with uranyl acetate (2%). Samples were observed at 200.0 k× magnification.

Mice and TC-1 Cell Lines
TC-1 cells (murine C57BL/6 lung epithelial cells transformed with HPV-16 E6/E7 and ras oncogenes) were obtained from TC Wu, John Hopkins Cervical Cancer Research Lab, USA. TC-1 cells were cultured and maintained at 37 • C/5% CO 2 in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum. Female C57BL/6 (6-8-weekold) mice were purchased from the Animal Resources Centre (Perth, Western Australia). Animal experiments were approved by The University of Queensland Animal Ethics Committee (UQDI/TRI/351/15 and UQDI/032/20) and were carried out in accordance with National Health and Medical Research Council (NHMRC) of Australia guidelines.

In Vivo Tumor Treatment Experiments
C57BL/6 Female mice (8 mice per group in pilot study, and 6 mice per group in the second study) were challenged subcutaneously in the flank with 2 × 10 5 TC-1 tumor cells/mouse (i.e., 1 × 10 5 TC-1 tumor cells/side). After 7 days (pilot) or 11 days (second study), mice were injected in the flank with the vaccine candidates. Each mouse received peptide (100 µL) or liposome formulation containing antigen (100 µg). Mice also received PBS (100 µL, 1×) as a negative control. L1 or a formulation containing 8Qm (100 µg) and CpG ODN 1826 adjuvant (20 µg) dissolved in PBS were used as a positive control. Each mouse received a single dose of the vaccine/control solution. Tumor size was measured (by palpation and calipers) and recorded for each mouse every 2 days. Tumor volume was calculated using the formula: V (cm 3 ) = 3.14 × [largest diameter × (perpendicular diameter)2]/6. The average tumor size per group (n = 8 or 6) was also calculated. Mice were euthanized when tumors reached 1 to 3 cm or if they started bleeding to avoid unnecessary suffering. An additional cohort of C57BL/6 mice (6 mice per group) were immunized with antigens, as described above. Mouse spleens were collected from these animals 10 days post immunization and used for the ELISPOT assay.

IFN-Gamma ELISPOT Assays
ELISPOT plates were coated with 5 µg/mL IFN-γ capture antibody (clone 14-7313-85 eBioscience, San Diego, CA, USA) in PBS at 4 • C overnight. The plates were then blocked with RPM/20% fetal bovine serum at room temperature for 3 h. Splenocytes from C57BL/6 mice were harvested from the spleens of naïve and immunized mice. The red blood cells were depleted using red blood cell lysing buffer (0.155 M ammonium chloride in 0.01 M Tris-HCl buffer, Sigma). Splenocytes were then resuspended in RPMI (Sigma) supplemented with 20% FACs (100 U/mL penicillin, 100 µg/mL streptomycin and 50 µM β-mercaptoethanol). The cells were plated at 5 × 10 5 cells per well in triplicate on ELISPOT plates. The E7 peptide (10 µg/mL) was added alongside 10 ng/mL rhIL-2 to a final volume of 200 µL per well. Plates were incubated for 8 h at 37 • C and washed. Biotinylated IFN-γ detection antibody (2 µg/mL; clone R4-6A2; eBioscience) in BSA (1%) was then added at room temperature. The plates were washed, and bound cytokine was visualized with 3-amino-9-ethylcarbozole. Spots were counted with an ELISPOT reader.

Statistical Analysis
All data were analyzed using GraphPad Prism 7 software. Kaplan−Meier survival curves were used to compare tumor treatment candidates. Differences in survival treatments were determined using the log-rank (Mantel-Cox) test, with p < 0.05 considered statistically significant. ELISPOT statistical analysis was performed using a one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test (ns, p > 0.05; *, p < 0.05).

Vaccine Design
In our previous attempts to develop efficient vaccine delivery systems, we have demonstrated that conjugation of the polyleucine moiety to a peptide antigen greatly enhanced antigen-specific humoral immune responses [20,21,[23][24][25][26][27]. We also found that the polymer-antigen conjugate D-8Qm once formulated into liposomes (L1) was able to eradicate, to some extent, seven-days-old cancer in mice [27]. Herein, we have replaced the polyacrylate dendrimer (D) with polyleucine to produce the self-assembling conjugate, pLeu-8Qm. In addition, we have also combined the polyleucine strategy with a liposomal formulation applied for D-8Qm (L1) to produce L2. To further improve vaccine efficacy, we anchored a variety of DC-targeting moieties to L2 liposomes (Figure 1), as antigen uptake by DCs (and other antigen presenting cells) is a key process in initializing natural immune responses, including anticancer cellular immunity [33]. All the targeting moieties have been lipidated to allow their anchoring to liposomes. Here, lipopeptides D1 and D2 were bearing DCpep-1 and DCpep-2 epitopes, respectively, where both DCpep-1 and DCpep-2 are reported to have DC-targeting properties [34]. In addition, DCpep-1 has been shown to stimulate specific T-cell responses against tumors [35][36][37], while DCpep-2 regulated inflammatory mediators [38,39]. The incorporation of a mannose-based ligand into liposome formulations has been previously reported as being effective in promoting CTL responses [40][41][42]. We designed our ligands (M1-M3) to have different spacer lengths between the mannose and lipidic moieties, where the importance of these spacers has been previously reported [3,[43][44][45]. We have also demonstrated the mechanism of mannosereceptor-mediated uptake [46]. Importantly, the ability of M2 anchored into liposomes to greatly enhance humoral immune responses against a bacterial antigen has also been proven (unpublished data). Along the same line, we also demonstrated that the mannoselipid conjugate M3 easily anchored into liposomes induced cellular immunity against malaria and babesia parasites [31,[47][48][49]. Finally, we had also previously proven that the lapidated-cell-penetrating peptide KALA (CPP), upon anchoring to liposomes, triggered the production of opsonic antibodies against a co-anchored peptide antigen [32]. The ability of cell-penetrating peptides to improve the cellular immunity of the co-delivered antigen has also been reported by other groups [50]. Identically produced cationic liposomes were used in all formulations (L1, L2, L2DC1, L2DC2, L2M1, L2M2, L2M3) except L2CPP, where neutral multilamellar liposomes were applied. Here, the use of cationic liposomes in combination with a CPP was redundant due to the very high cationic charge of the KALA peptide, and a CPP was more effective when combined with multilamellar (not unilamellar) liposomes [32].

Vaccine Preparation
All the peptide components of vaccines and targeting moieties were synthesized using Fmoc-SPPS. pLeu-8Qm was also produced as a single molecule using the standard SPPS methodology. pLeu-8Qm was incorporated in DC-targeting liposomal formulations and used for a pilot immunization study. The polymeric conjugate D-8Qm was produced by conjugation of the 8Qm peptide to polyacrylate D by the copper-catalyzed alkyne−azide cycloaddition (CuAAC) reaction and self-assembled into particles by the solvent replacement method, as previously reported [19,51]. Dialysis was performed in water for three days to remove residual peptide, copper and organic solvents, and elemental analysis was used to confirm the formation of the product, which was in line with the methods of many previous studies [10,11,20,51,52], as the conjugate contained a higher nitrogen/carbon ratio compared with the polyacrylate alone. Theoretical and observed (nitrogen [N]/carbon [C]) ratios were used to calculate the exact substitution of the polymer core with the peptide epitopes. The observed nitrogen-carbon ratio for D-8Qm (N/C = 0.115) was higher than that of the polymer alone (N/C = 0.017) and corresponded to 83% of the substitution rate of the dendrimer with 8Qm peptide (similar to that previously reported at 85% [19]). Both D-8Qm and pLeu-8Qm, as amphiphilic conjugates, were easily incorporated (anchored) into liposomes [19,21] to form L1 and L2, respectively (Figure 1).
The size and surface charge of the vaccine candidates were analyzed using DLS (Table 1, Supporting Information, Figure S5). Both D-8Qm and pLeu-8Qm produced aggregates of sub/micron sizes with high polydispersity (polydispersity index; PDI = 0.6-0.7) and a similar negative charge (ζ = −10-−14 mV). In contrast, all liposome formulations were positively charged and relatively monodispersed, except L1 and L2CPP. Unilamellar liposomes bearing pLeu-8Qm were produced in the size range of 150-200 nm, which was typically more immunogenic compared with larger-sized particles [54][55][56]. Multilamellar liposome L2CPP formed submicron particles (0.6 µm, PDI = 0.4) with a high positive charge. Additionally, the size and morphology of the vaccine candidates were analyzed by TEM (Supporting Information, Figure S6). Large particle aggregates were detected on images of D-8Qm, while all liposomes formed typical spherical liposome structures, which were uniform in size (100-250 nm) without visible traces of large aggregates, as detected by DLS in the case of L1 and L2DC1 (Supporting Information, Figure S5).

Immunological Evaluation of the Vaccine Candidates
At first, the therapeutic effect of the vaccine candidates on established tumors was evaluated in eight-week-old female C57BL/6 mice seven days post TC-1 tumor implantation ( Figure 2). Average tumor growth was fast, with only 25% of the negative control (PBS-treated) mice surviving to day 21. Alone, pLeu-8Qm decreased tumor growth and prolonged mouse survival. The incorporation of pLeu-8Qm into liposomes (L2) greatly improved vaccine efficacy. While all mice treated with L2 survived 64 days, two of the eight mice had tumor growth recurrence after days 34 and 56. The improved ability of the liposome-based L2 vaccine candidate in triggering an antitumor immune response compared with pLeu-8Qm alone might be attributed to the size of the particles. pLeu-8Qm formed highly polydisperse submicroparticles, while L2 formed nanoparticles (<200 nm); however, smaller particles are widely reported to be more immunogenic [57,58].

Immunological Evaluation of the Vaccine Candidates
At first, the therapeutic effect of the vaccine candidates on established tumors was evaluated in eight-week-old female C57BL/6 mice seven days post TC-1 tumor implantation ( Figure 2). Average tumor growth was fast, with only 25% of the negative control (PBS-treated) mice surviving to day 21. Alone, pLeu-8Qm decreased tumor growth and prolonged mouse survival. The incorporation of pLeu-8Qm into liposomes (L2) greatly improved vaccine efficacy. While all mice treated with L2 survived 64 days, two of the eight mice had tumor growth recurrence after days 34 and 56. The improved ability of the liposome-based L2 vaccine candidate in triggering an antitumor immune response compared with pLeu-8Qm alone might be attributed to the size of the particles. pLeu-8Qm formed highly polydisperse submicroparticles, while L2 formed nanoparticles (<200 nm); however, smaller particles are widely reported to be more immunogenic [57,58].  The survival rate of each group was compared with the negative control (PBS) and was analyzed using the log-rank (Mantel-Cox) test (*** p < 0.001; **** p < 0.0001). (e) Assessment of CD8+ T-cell responses in mice subcutaneously immunized with the vaccine candidates. Mouse spleens were harvested ten days after vaccination and IFN-γ production in response to short E749-57 (RAHYNIVTF) CD8 peptide epitope was determined by ELISPOT (n = 6 mice/group). The L3 group (irrelevant control) was identical to L2 except the antigen 8Qm was replaced with J8 B-cell epitope derived from group A streptococcus (unrelated epitope). ELIPOST data were pooled from two independent experiments and analyzed using one-way ANOVA followed by Tukey's post hoc multiple comparison test (ns, p > 0.05; *, p < 0.05).
As the vaccine candidates investigated in this study aimed to stimulate CD8+ CTLs in order to eradicate HPV-infected cells, the recall of IFN-γ production by CD8+ T-cells was examined in immunized mice in response to MHC classIrestricted E7 peptide re-stimulation by ELISPOT (Figure 2). The immunization of mice with pLeu-8Qm delivered by liposomes induced significantly higher levels of IFN-γ compared with PBS, corresponding with its antitumor potency. This demonstrated that the downstream priming of the T-cells via INF-γ activation led to the eradication of tumors following subcutaneous immunization. Moreover, this antitumor efficacy was achieved without the presence of an external adju-vant, assistance of chemotherapy, multiple immunizations and/or boosting with immune checkpoint inhibitors [59][60][61][62].
Encouraged by the outcomes of the pilot study, we designed several new vaccine candidates ( Figure 1) and examined them under more demanding conditions (Figure 3, Supporting Information, Figure S7). The efficacy of the vaccine candidates was evaluated by testing against 11-day-old tumors following a single immunization to demonstrate the superiority of the strategy compared with our previous studies. These previous studies utilized 8Qm-based peptide-polymer conjugate vaccines targeting 3-day-old smaller tumors [11], a prime-boost immunization schedule using HPV E6-and E7-epitope-based multivalent conjugates with a booster [18], or the use of 8Qm emulsified in the commercialized adjuvant, Montanide ISA 51, to achieve significant antitumor effects [10]. Previously examined polyacrylate-based vaccine D-8Qm and its liposomal formulation L1 have also been assessed. In addition, the classical "cellular" adjuvant, CpG, was used as a positive control. CpG has been recently extensively investigated as an effective immune stimulator in the development of therapeutic vaccines against HPV-related cancers [4,7,[63][64][65]. Three vaccine candidates, L2, L2DC1 and L2M2, delayed tumor regrowth and induced tumor regression with the highest survival rate (67% on day 60). All surviving mice treated with L2 achieved complete tumor regression on day 60, while full tumor regression was observed in three out of six mice in both L2DC1 and L2M2 ( Figure 3). Moreover, L2M2 induced some tumor regression in all mice, and these mice survived up to day 47 following a single immunization. Importantly, all three vaccines (L2, L2DC1 and L2M2) were significantly more effective as therapeutic vaccines than 8Qm adjuvanted with CpG. Mice immunized with 8Qm/CpG showed a delay in tumor growth compared with the negative control (PBS); however, they ultimately had the same survival rate of 0% on day 60. Among the three leading formulations, even the simple liposome formulation L2 was more effective at inducing tumor regression than the previous leading vaccine candidates, D-8Qm and L1 [19]. It is also worth noting that when unconjugated and adjuvant-free 8Qm was incorporated into the same liposomal formulation, therapeutic potential was not observed [19]. Other DC-targeting formulations (L2DC2, L2M1, L2M3 and L2CPP) were less effective than the untargeted L2 formulation and not significantly more effective than 8Qm/CpG. In addition, there was no correlation between the size of liposome particles and vaccine efficacy. The leading vaccine candidates, L2, L2DC1 and L2M2, had particle sizes in similar ranges to other liposomal formulations, except L2CPP (Table 1).
Notably, targeting moieties did not improve the final survival rate; however, the formulation bearing lipidated mannose (M2) delayed tumor growth more significantly than other formulations. Thus, further investigations on this formulation are required. For example, the anticancer efficacy of L2M2 might be greatly enhanced by the administration of a boosting dose around 3-4 weeks following the primary vaccination.
In our study, mice immunized with L2, L2DC1 and L2M2 demonstrated improved survival 60 days after the inoculation of the same density of TC1 tumor cells (1 × 10 5 ), despite the more advanced stage of tumor development at the time of the initiation of vaccine treatment (11 days post tumor cell inoculation versus 7 or 8 days in other studies [66,67]). Furthermore, within the framework of a single immunization schedule, the L2-, L2DC1-and L2M2-immunized mice displayed tumor eradication in some cases, outperforming prior studies utilizing multiple immunizations, which failed to achieve complete tumor regression [66][67][68]. Additionally, the efficacy of our vaccine formulation was maintained without the use of multiepitopes from both E6 and E7 proteins [69]; the addition of adjuvants, such as flagellin [70], Montanide ISA 720 [71] or a combination of poly I:C and CpG [63]; or synergistic therapy with immune checkpoint inhibitors, such as anti-CTLA4 [72] or anti-PD1 [73].  Notably, targeting moieties did not improve the final survival rate; however, the formulation bearing lipidated mannose (M2) delayed tumor growth more significantly than other formulations. Thus, further investigations on this formulation are required. For example, the anticancer efficacy of L2M2 might be greatly enhanced by the administration of a boosting dose around 3-4 weeks following the primary vaccination.
In our study, mice immunized with L2, L2DC1 and L2M2 demonstrated improved survival 60 days after the inoculation of the same density of TC1 tumor cells (1 × 10 5 ), despite the more advanced stage of tumor development at the time of the initiation of vaccine treatment (11 days post tumor cell inoculation versus 7 or 8 days in other studies [66,67]). Furthermore, within the framework of a single immunization schedule, the L2-, L2DC1-and L2M2-immunized mice displayed tumor eradication in some cases, outperforming prior studies utilizing multiple immunizations, which failed to achieve complete tumor regression [66][67][68]. Additionally, the efficacy of our vaccine formulation was maintained without the use of multiepitopes from both E6 and E7 proteins [69]; the addition of adjuvants, such as flagellin [70], Montanide ISA 720 [71] or a combination of poly I:C and CpG [63]; or synergistic therapy with immune checkpoint inhibitors, such as anti-CTLA4 [72] or anti-PD1 [73].

Conclusions
We demonstrated that a fully defined, natural, hydrophobic amino-acid-based polymer conjugated to peptide antigen acts as an efficient vaccine delivery system. Importantly, polyleucine mimics the transmembrane fragments of proteins, allowing the anchoring of the conjugate to liposomes and effectively triggering cellular immunity to destroy tumor cells following a single immunization without the use of an additional adjuvant. In contrast with nanotechnology-based strategies, which usually offer incompletely defined systems, our strategy utilizes a polymer built from natural amino acids that is

Conclusions
We demonstrated that a fully defined, natural, hydrophobic amino-acid-based polymer conjugated to peptide antigen acts as an efficient vaccine delivery system. Importantly, polyleucine mimics the transmembrane fragments of proteins, allowing the anchoring of the conjugate to liposomes and effectively triggering cellular immunity to destroy tumor cells following a single immunization without the use of an additional adjuvant. In contrast with nanotechnology-based strategies, which usually offer incompletely defined systems, our strategy utilizes a polymer built from natural amino acids that is both a single molecule and a single isomer. Given the need for the vaccine treatment of intracellular infectious diseases and malignancies, we anticipate numerous applications for the vaccine delivery system presented here.